Arenaviruses are responsible for severe hemorrhagic fevers with high morbidity and mortality worldwide. There are currently no vaccines or specific therapies against arenavirus infection and these viruses are thus recognized as Category A priority pathogens for antiviral research. Therapeutic strategies aimed at preventing virus entry into the host cell hold promise for combating these lethal infections. Arenaviruses enter the cell through pH-dependent fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein (GPC). Unlike other Class I viral fusion proteins, each protomer in the trimeric GPC complex contains three subunits: GP1, GP2 and a stable signal peptide (SSP). Our studies have demonstrated that SSP plays a fundamental role in controlling pH-induced activation of arenavirus membrane fusion and its inhibition by lead small-molecule compounds. We find that the short external loop of the membrane-embedded SSP hairpin acts in conjunction with the GP2 fusion subunit to sense acidic pH in the maturing endosome and thereby trigger the structural transitions leading to virus-cell fusion. This fusion-activation function is also regulated by specific interactions between the membrane-spanning segments of the two subunits. By characterizing intersubunit interactions in the virion membrane, we aim to elucidate the structural and mechanistic basis for arenaviral pH sensing and fusion activation. In addition, we will determine how interactions between the transmembrane elements of GPC contribute to arenavirus morphogenesis. The specific aims of this proposal are to: (1) Determine the spatial organization of the SSP and GP2 transmembrane segments by oxidative cysteine crosslinking and identify amino-acid residues critical for GPC membrane fusion and its inhibition. Preliminary genetic and modeling studies suggest that the first membrane-spanning region of SSP (TM1) and the transmembrane domain (TMD) of GP2 each form amphipathic and functionally asymmetric ?-helices that interact to modulate arenaviral membrane fusion. We will determine the physical arrangement of these segments in GPC by combined cysteine-scanning mutagenesis and oxidative crosslinking. We will then use alanine replacements to characterize the role of the intersubunit contacts in GPC biosynthesis, pH-dependent fusion activity and sensitivity to small-molecule fusion inhibitors. (2) Characterize the structure and function of the membrane-spanning domain of GPC in the intact virion to elucidate roles in virus entry and its inhibition, as well as in virion assembly and budding. We will assess the pattern of oxidative crosslinking in Junin Candid#1 viruses generated by reverse genetics to determine whether the membrane-spanning domain of GPC is altered on virion assembly, inhibitor binding or exposure to acidic pH. We will characterize the impact of specific intersubunit contacts on virus growth, specific infectivity, sensitivity to acidic pH and fusion inhibitors, and on virion assembly, budding and protein composition. These latter experiments may identify novel roles for GPC in virion morphogenesis.